Direct identification of ubiquitination sites on ubiquitin-conjugated CHIP using MALDI mass spectrometry

Dongxia Wang, Wanping Xu, Sara C. McGrath, Cam Patterson, Len Neckers, Robert J. Cotter

Research output: Contribution to journalArticlepeer-review

26 Scopus citations


The study of protein ubiquitination, a post-translational modification by ubiquitin, has emerged as one of the most active areas in biology because of the important role of this type of modification on the regulation of various cellular proteins. Advances in techniques for the determination and site mapping of protein ubiquitination can facilitate the elucidation of molecular mechanisms of this modification. We have recently described a novel method for identifying peptides containing ubiquitinated amino acid residues, based on the MALDI-MS/MS analysis of tryptic peptide derivatives. In particular, we have utilized N-terminal sulfonation of these peptides to provide a unique fragmentation pattern that leads to the direct identification and sequencing of ubiquitin modified peptides. Here we present an application of this new method on the characterization of ubiquitin conjugated C-terminal Hsc70-interacting protein (CHIP), a recently identified U-box containing E3 enzyme. Three peptides bearing ubiquitination sites have been identified from the digest of ubiquitinated CHIP; one of these was a site on CHIP, while the other two were found on the ubiquitin molecules, demonstrating that sulfonation of tryptic peptides is a general and efficient method for characterizing protein ubiquitination.

Original languageEnglish (US)
Pages (from-to)1554-1560
Number of pages7
JournalJournal of proteome research
Issue number5
StatePublished - Sep 2005


  • CHIP
  • MALDI mass spectrometry
  • N-terminal sulfonation
  • Ubiquitination

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)


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